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- PDB-8rch: CryoEM structure of mTORC1 with a paediatric kidney cancer-associ... -

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Basic information

Entry
Database: PDB / ID: 8rch
TitleCryoEM structure of mTORC1 with a paediatric kidney cancer-associated 1455-EWED-1458 duplication in mTOR, overall refinement
Components
  • Eukaryotic translation initiation factor 4E-binding protein 1
  • Regulatory-associated protein of mTOR
  • Serine/threonine-protein kinase mTOR
  • Target of rapamycin complex subunit LST8
KeywordsSIGNALING PROTEIN / Target of Rapamycin / mTOR / Cancer-associated mutants
Function / homology
Function and homology information


RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / eukaryotic initiation factor 4E binding / TORC2 complex ...RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / eukaryotic initiation factor 4E binding / TORC2 complex / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / regulation of membrane permeability / heart valve morphogenesis / negative regulation of lysosome organization / nucleus localization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / calcineurin-NFAT signaling cascade / regulation of osteoclast differentiation / voluntary musculoskeletal movement / TORC1 signaling / positive regulation of odontoblast differentiation / positive regulation of keratinocyte migration / cellular response to L-leucine / Amino acids regulate mTORC1 / MTOR signalling / cellular response to nutrient / cellular response to methionine / Energy dependent regulation of mTOR by LKB1-AMPK / regulation of autophagosome assembly / energy reserve metabolic process / negative regulation of cell size / ruffle organization / cellular response to osmotic stress / positive regulation of osteoclast differentiation / protein serine/threonine kinase inhibitor activity / enzyme-substrate adaptor activity / anoikis / cardiac muscle cell development / negative regulation of protein localization to nucleus / positive regulation of transcription by RNA polymerase III / negative regulation of calcineurin-NFAT signaling cascade / regulation of myelination / regulation of cell size / negative regulation of macroautophagy / positive regulation of oligodendrocyte differentiation / positive regulation of actin filament polymerization / lysosome organization / Macroautophagy / positive regulation of myotube differentiation / protein kinase activator activity / oligodendrocyte differentiation / Constitutive Signaling by AKT1 E17K in Cancer / mTORC1-mediated signalling / germ cell development / behavioral response to pain / : / CD28 dependent PI3K/Akt signaling / social behavior / HSF1-dependent transactivation / positive regulation of TOR signaling / neuronal action potential / response to amino acid / TOR signaling / 'de novo' pyrimidine nucleobase biosynthetic process / regulation of macroautophagy / positive regulation of G1/S transition of mitotic cell cycle / positive regulation of translational initiation / cellular response to nutrient levels / endomembrane system / positive regulation of lamellipodium assembly / phosphorylation / positive regulation of lipid biosynthetic process / phagocytic vesicle / positive regulation of epithelial to mesenchymal transition / heart morphogenesis / cardiac muscle contraction / regulation of cellular response to heat / translation repressor activity / positive regulation of stress fiber assembly / 14-3-3 protein binding / negative regulation of translational initiation / cytoskeleton organization / positive regulation of endothelial cell proliferation / T cell costimulation / translation initiation factor binding / cellular response to amino acid starvation / positive regulation of glycolytic process / positive regulation of mitotic cell cycle / cellular response to starvation / protein serine/threonine kinase activator activity / negative regulation of autophagy / response to nutrient / response to nutrient levels / post-embryonic development / VEGFR2 mediated vascular permeability / regulation of signal transduction by p53 class mediator / positive regulation of peptidyl-threonine phosphorylation
Similarity search - Function
Eukaryotic translation initiation factor 4E binding / Eukaryotic translation initiation factor 4E binding protein (EIF4EBP) / Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / Domain of unknown function ...Eukaryotic translation initiation factor 4E binding / Eukaryotic translation initiation factor 4E binding protein (EIF4EBP) / Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / Rapamycin binding domain / HEAT repeat / HEAT repeat / : / FATC domain / PIK-related kinase, FAT / FAT domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Quinoprotein alcohol dehydrogenase-like superfamily / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Serine/threonine-protein kinase mTOR / Eukaryotic translation initiation factor 4E-binding protein 1 / Regulatory-associated protein of mTOR / Target of rapamycin complex subunit LST8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsAnandapadamanaban, M. / Hay, I.M. / Perisic, O. / Williams, R.L.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Cancer Research UKDRCPGM 100014 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_U105184308 United Kingdom
European Molecular Biology Organization (EMBO)EMBO ALTF 603-2019European Union
CitationJournal: To Be Published
Title: cryoEM structure of mTORC1 with the cancer-associated 1455-EWED-1458 duplication variant, overall refinement
Authors: Anandapadamanaban, M. / Hay, I. / Perisic, O. / Williams, R.
History
DepositionDec 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine/threonine-protein kinase mTOR
D: Target of rapamycin complex subunit LST8
W: Regulatory-associated protein of mTOR
Z: Eukaryotic translation initiation factor 4E-binding protein 1
B: Serine/threonine-protein kinase mTOR
E: Target of rapamycin complex subunit LST8
Y: Regulatory-associated protein of mTOR
X: Eukaryotic translation initiation factor 4E-binding protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)975,03214
Polymers973,9228
Non-polymers1,1106
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 8 molecules ABDEWYZX

#1: Protein Serine/threonine-protein kinase mTOR / FK506-binding protein 12-rapamycin complex-associated protein 1 / FKBP12-rapamycin complex- ...FK506-binding protein 12-rapamycin complex-associated protein 1 / FKBP12-rapamycin complex-associated protein / Mammalian target of rapamycin / mTOR / Mechanistic target of rapamycin / Rapamycin and FKBP12 target 1 / Rapamycin target protein 1


Mass: 289257.969 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTOR, FRAP, FRAP1, FRAP2, RAFT1, RAPT1 / Production host: Homo sapiens (human)
References: UniProt: P42345, non-specific serine/threonine protein kinase
#2: Protein Target of rapamycin complex subunit LST8 / TORC subunit LST8 / G protein beta subunit-like / Protein GbetaL / Mammalian lethal with SEC13 ...TORC subunit LST8 / G protein beta subunit-like / Protein GbetaL / Mammalian lethal with SEC13 protein 8 / mLST8


Mass: 35910.090 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MLST8, GBL, LST8 / Production host: Homo sapiens (human) / References: UniProt: Q9BVC4
#3: Protein Regulatory-associated protein of mTOR / Raptor / p150 target of rapamycin (TOR)-scaffold protein


Mass: 149200.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPTOR, KIAA1303, RAPTOR / Production host: Homo sapiens (human) / References: UniProt: Q8N122
#4: Protein Eukaryotic translation initiation factor 4E-binding protein 1 / 4E-BP1 / eIF4E-binding protein 1 / Phosphorylated heat- and acid-stable protein regulated by ...4E-BP1 / eIF4E-binding protein 1 / Phosphorylated heat- and acid-stable protein regulated by insulin 1 / PHAS-I


Mass: 12592.968 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EIF4EBP1 / Production host: Homo sapiens (human) / References: UniProt: Q13541

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Non-polymers , 2 types, 6 molecules

#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: mTORC1 complex is a dimer of a heterotrimeric complex including mTOR, RAPTOR and mLST8
Type: COMPLEX
Details: This cancer-associated mTORC1 variant consists of mTOR 1455-EWED-1458 duplication, RAPTOR and mLST8 complex and its bound to a substrate 4EBP1.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 1.09 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: EPU / Category: image acquisition
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264890 / Algorithm: FOURIER SPACE / Details: RELION was used for final reconstructions. / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 122.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: Initial local fitting was done in Coot and Chimera and then subjected to ROSETTA for model rebuilding. Finally, in Phenix real-space refine was performed for the final model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 122.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00450668
ELECTRON MICROSCOPYf_angle_d0.716968727
ELECTRON MICROSCOPYf_chiral_restr0.04397815
ELECTRON MICROSCOPYf_plane_restr0.00668737
ELECTRON MICROSCOPYf_dihedral_angle_d6.24436708

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